The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

IL-28B is a Key Regulator of B- and T-Cell Vaccine Responses against Influenza

Influenza is a major cause of morbidity and mortality in immunosuppressed persons, and vaccination often confers insufficient protection. IL-28B, a member of the interferon (IFN)-λ family, has variable expression due to single nucleotide polymorphisms (SNPs). While type-I IFNs are well known to modulate adaptive immunity, the impact of IL-28B on B- and T-cell vaccine responses is unclear. Here we demonstrate that the presence of the IL-28B TG/GG genotype (rs8099917, minor-allele) was associated with increased seroconversion following influenza vaccination (OR 1.99 p = 0.038). Also, influenza A (H1N1)-stimulated T- and B-cells from minor-allele carriers showed increased IL-4 production (4-fold) and HLA-DR expression, respectively. In vitro, recombinant IL-28B increased Th1-cytokines (e.g. IFN-γ), and suppressed Th2-cytokines (e.g. IL-4, IL-5, and IL-13), H1N1-stimulated B-cell proliferation (reduced 70%), and IgG-production (reduced>70%). Since IL-28B inhibited B-cell responses, we designed antagonistic peptides to block the IL-28 receptor α-subunit (IL28RA). In vitro, these peptides significantly suppressed binding of IFN-λs to IL28RA, increased H1N1-stimulated B-cell activation and IgG-production in samples from healthy volunteers (2-fold) and from transplant patients previously unresponsive to vaccination (1.4-fold). Together, these findings identify IL-28B as a key regulator of the Th1/Th2 balance during influenza vaccination. Blockade of IL28RA offers a novel strategy to augment vaccine responses.     

The Role of Hormones in the Differences in the Incidence of Breast Cancer between Mongolia and the United Kingdom

Background

There are striking differences in breast cancer incidence between Asian and western women. Rates vary substantially within Asia also, with Mongolia''s even lower than China''s. These profound differences have been speculated to be due in part to diet, mediated by circulating hormone concentrations.

Methods

Sex steroid hormone concentrations were measured in women living in Ulaanbaatar, Mongolia and the United Kingdom (U.K.). Diet was obtained by interview and national survey data. Mean hormone differences were compared by country, and systematic variation by number of days since last menstrual period was modeled and adjusted for age and parity; difference in overall area under the curves was assessed.

Findings

The diet in Mongolia was higher in meat and dairy than in the U.K. Mean testosterone concentrations were 18.5% lower (p<0.0001) while estradiol concentrations were 19.1% higher (p = 0.02) in Mongolian than British women, adjusted for age and parity. Progesterone was almost 50% higher in Mongolian women (p = 0.04), particularly during the follicular phase and early luteal surge. Hormone concentrations generally were similar in Mongolian women born in Ulaanbaatar compared with those born in rural areas, although there was a decreasing progesterone trend by degree of westernization (rural Mongolia; urban Mongolia; U.K.). Mean hormone differences were similar when restricted to parous women, and with further adjustment for body mass index, height, and smoking status.

Interpretation

These data augment accumulating evidence that circulating estrogens are unlikely to explain reduced breast cancer rates in Asia compared with the west, and suggest casting a wider net with respect to biomarkers. Lower testosterone and higher progesterone in Mongolian women raise the possibility that these hormones may be important to consider. In addition, the almost exclusive dietary reliance of Mongolians on meat and dairy argues against beneficial effects of a low-fat diet on circulating hormones explaining international breast cancer differences.     

p53/TAp63 and AKT Regulate Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling through Two Independent Parallel Pathways in the Presence of DNA Damage

Under conditions of DNA damage, the mammalian target of rapamycin complex 1 (mTORC1) is inhibited, preventing cell cycle progression and conserving cellular energy by suppressing translation. We show that suppression of mTORC1 signaling to 4E-BP1 requires the coordinated activity of two tumor suppressors, p53 and p63. In contrast, suppression of S6K1 and ribosomal protein S6 phosphorylation by DNA damage is Akt-dependent. We find that loss of either p53, required for the induction of Sestrin 1/2, or p63, required for the induction of REDD1 and activation of the tuberous sclerosis complex, prevents the DNA damage-induced suppression of mTORC1 signaling. These data indicate that the negative regulation of cap-dependent translation by mTORC1 inhibition subsequent to DNA damage is abrogated in most human cancers.     

Occurrence of proteins immunoreactive with anti-coupling factor B in phosphorylating membrane preparations

Coupling factor B has been isolated from beef heart mitochondria, apparently in multiple forms which differ in molecular weight and specific activity. Since it has no known intrinsic catalytic activity, detection and quantitation have been based upon the factor B-dependent stimulation of ATP-linked activities in factor B-deficient submitochondrial particles. This communication reports the development of a reliable and more universally applicable enzyme-linked immunosorbent assay (ELISA) for detection and quantitation of factor B in soluble or membranous preparations. The assay requires nanoliter volumes of rabbit antiserum raised against purified factor B and will detect nanogram amounts of the coupling factor. Analysis of beef heart submitochondrial particles using a competitive binding ELISA indicated a factor B content of 0.27 nmol/mg protein, making factor B stoichiometric with F₁ (0.3–0.6 nmol/mg). Furthermore, application of the factor B ELISA has indicated the presence of material cross-reacting with the beef heart factor B-antiserum in phosphorylating membranes from chloroplasts, Escherichia coli, Paracoccus denitrificans and the thermophilic bacterium, PS3. Negative results were obtained with mitochondria and microsomes from rat liver, purple membranes from Halobium halobacterium and sarcoplasmic reticulum from rabbit skeletal muscle.     

Homophilic binding of mouse monoclonal antibodies against GD3 ganglioside.

R24, a mouse IgG3 mAb against GD3 ganglioside, was shown to bind to itself in a homophilic manner. This was demonstrated by augmented binding of 125I-labeled R24 to the cell surface of GD3+ cells by unlabeled R24 and by direct binding of biotinylated R24 to R24 adsorbed on solid phase. Although homophilic binding was evident when R24 was bound to solid phase, R24-R24 aggregates could not be detected in solution under otherwise identical conditions. R24 bound to four other mAb (two IgG3, one IgG2a, one IgM) directed against GD3 but did not bind to a panel of 21 other mAb including other IgG3 mAb and mAb directed against non-GD3 ganglioside. Evidence implicating the GD3-binding site of R24 in homophilic binding included the following observations: 1) F(ab')2 fragments of R24 could bind to R24, 2) an antiidiotypic mAb against the GD3-binding site of R24 inhibited R24 homophilic binding, 3) an IgM anti-GD3 mAb also demonstrated homophilic binding to R24, and 4) homophilic binding was a function of immunoreactivity and avidity for GD3. R24 variants with 40-fold lower avidity for GD3 demonstrated a similar decrease in homophilic binding. Inasmuch as R24 bound to R24 F(ab')2 fragments and specifically to anti-GD3 mAb, it appeared that the target for homophilic binding was an epitope within the V region of anti-GD3 mAb. It is likely that homophilic interactions result in increased affinity of R24 for GD3 through increased effective valency of antibody-Ag complexes.